Miniaturization and Optimization of a Two-Plasmid Expression Pathway in myTXTL® Cell-Free Protein Expression Kit (Arbor Biosciences) Using Labcyte Echo® Acoustic Technology

Evelyn Eggenstein1, Jared Bailey2, Jean-Marie Rouillard1, John Lesnick2

1Arbor Biosciences, Ann Arbor MI, USA, 2Labcyte Inc. San Jose CA, USA

INTRODUCTION

In vitro transcription (TX)-translation

(TL) is a rapidly developing technol-

ogy with vast potential for synthetic

biology and bio-engineering.

myTXTL® (FIG. 1), an E. coli based

cell-free protein expression system

entirely relying on the endogenous

TXTL machinery, is a versatile and all-

in-one solution for many applica-

tions, such as the development of

multi-stage gene circuits, and

screening of complex protein

libraries. For both examples, high

sample throughput at an affordable

cost is desirable.

HIGHLIGHTS• Echo® 525 Liquid Handler reproducibly and reliably transfers

myTXTL® Master Mix.

• Miniaturization reduces overall assay costs by a factor ofthree due to lowered reagent consumption.

• The speed of arraying allows for time-dependent reads ofmultiple plasmid inputs in a myTXTL® reaction.

EXPERIMENTAL DESIGN

A typical myTXTL® reaction has a total volume of 12 μL and

consists of a ready-to-use myTXTL® Master Mix and a nucleotide

template. Here, we pursued cell-free expression of a model pro-

tein (eGFP mutant) under transcriptional control of the T7 system.

This requires the presence of T7 RNA polymerase, which was

supplied to the myTXTL® reaction encoded on a plasmid (FIG 4).

FIG 1. myTXTL® workflow.

The Labcyte Echo® 525 Liquid Handler

(FIG. 2) allows rapid, accurate and

contact-free transfer of volumes at a

nanoliter scale with acoustic sound.

Its integrated Dynamic Fluid Analysis

technology enables the Echo® system

to easily adapt to various types of

fluid without the need for calibration,

which makes it ideal for handling

multi-component solutions (FIG 3).

This simplifies experimental setup and

enables a maximum degree of

flexibility for study design.

FIG 3. Acoustic droplet ejection (ADE) technology of the Echo® Liquid Handler.

Combining these two sophisticated platforms provides a

comprehensive solution for fully-automated high-throughput in

vitro protein expression: from template preparation to product

analysis. In this study, we demonstrate the ability to process Arbor

Biosciences‘ myTXTL® system in very low volumes and to

accelerate DNA titration and dispensing using the Labcyte Echo®

Liquid Handler. This method will lower overall cost by reducing

assay time and reagent consumption, while increasing experi-

mental complexity and minimizing the risk of contamination and

human errors.

FIG 2. Labcyte Echo® 525.

RESULTS

Assay 1. Miniaturization of myTXTL® reaction

In the course of generating a standard curve for quantification of

deGFP produced in vitro, the Echo® 525 Liquid Handler was able to

accurately dispense a deGFP standard at different concentrations

for volumes between 1 μL and 12 μL (FIG 5).

FIG 4. In vitro protein production in myTXTL® using the T7 expression system.

FIG 5. deGFP standard curve set up by acoustic transfer. deGFP fluorescence in the384-well plate was acquired in a PHERAstar® reader at 485 nm (ex) and 520 nm (em).

Assay 2. Optimization of a two-plasmid myTXTL® reaction

Using acoustic liquid transfer, reagents were arrayed into 384-well

plates to allow multiplexing of the dual-plasmid circuit (TABLE 1).

Then, plates were incubated at 29 °C using a Labcyte MicroClime®

lid to prevent evaporation, and fluorescence signal of deGFP

produced in myTXTL® was continuously recorded over 16 hours.

TABLE 1. Experimental scheme for evaluating a two-plasmid myTXTL ® reaction usingEcho® 525 Liquid Handler for reagent transfer.

Maximum production of deGFP was observed in the presence of

4.5 nM T7p14-deGFP and 37.5 pmol P70a-T7rnap (FIG 6). At

higher concentration of either plasmid, total deGFP yield was

reduced. Alterations in the concentration of P70a-T7rnap were

most pronounced over the time course of the experiment (FIG 7).

DISCUSSION

Arbor Biosciences’ myTXTL® Master Mix combined with the

Labcyte Echo® gives the ability to obtain fluorescent protein

readout from a multivariable DNA input within a matter of hours.

Rapid arraying, in combination with the reduced DNA and reagent

demand, allows for a large increase in the number of possible

experimental conditions as seen in Table 1. More work with

acoustically arrayed myTXTL® will be performed to further

highlight the flexibility of both systems in building a biological

circuit.

FIG 6. deGFP expression in myTXTL® as a function of plasmid concentration. EitherT7p14-deGFP (blue) or P70a-T7rnap (purple) were kept constant, while the con-centration of the corresponding plasmid was titrated over several magnitudes. Datarecorded after 4 h (circle), 4.5 h (square), 5 h (triangle), 5.5 h (diamond) and 6 h (in-verted triangle) is plotted.

FIG 7. Kinetic of deGFP production in myTXTL® under three different reaction con-ditions of the dual-plasmid system. The 4-6 h window is marked with dotted lines.Fluorescence signal was acquired every 5 min.

deGFP StandardmyTXTL® Master Mix

T7p14-deGFPP70a-T7rnap

diH2O

nLnL

nLnL

nL

50-1000

25-60025-400

30000-950